1. Field of the Invention
The present invention relates to a nucleic acid encoding the enzyme hyaluronate synthase, and to the use of this nucleic acid in the preparation of recombinant cells for the production of the hyaluronate synthase enzyme and hyaluronic acid. Hyaluronate is also known as hyaluronic acid or hyaluronan.
2. Description of the Related Art
The incidence of streptococcal infections is a major health and economic problem worldwide, particularly in developing countries (Rotta, 1988). One reason for this is due to the ability of Streptococcal bacteria to grow undetected by the body""s phagocytic cells (i.e., macrophages and polymorphonuclear cells (PMNs). These cells are responsible for recognizing and engulfing foreign microorganisms. One effective way the bacteria evade surveillance is by coating themselves with polysaccharide capsules, such as hyaluronic acid (HA) capsules. (Kendall et al., 1937). Since HA is generally nonimmunogenic (Quinn and Singh, 1957) , the encapsulated bacteria do not elicit an immune response and are, therefore, not targeted for destruction. Moreover, the capsule exerts an antiphagocytic effect on PMNs in vitro (Hirsch, et al., 1960) and prevents attachment of Streptococcus to macrophages (Whitnack, et al., 1981). Precisely because of this, in group A and group C Streptococci, the HA capsules are major virulence factors in natural and experimental infections (Kass, et al., 1944: Wessels, et al., 1991). Group A Streptococcus are responsible for numerous human diseases including pharyngitis, impetigo, deep tissue infections, rheumatic fever and a toxic shock-like syndrome (Schaechter, et al., 1989). The group C Streptococcus equisimilis is responsible for osteomyelitis (Barson, 1986), pharyngitis (Benjamin, et al., 1976) , brain abscesses (Dinn, 1971), and pneumonia (Rizkallah, et al., 1988: Siefkin, et al., 1983).
Structurally, HA is a high molecular weight linear polysaccharide of repeating disaccharide units consisting of N-acetylglucosamine (GlcNAc) and glucuronic acid (GlcA) (Laurent and Fraser, 1992). HA is the only glycosaminoglycan synthesized by both mammalian and bacterial cells, particularly groups A and C Streptococci. Some Streptococcus strains make HA which is secreted into the medium as well as HA capsules. The mechanism by which these bacteria synthesize HA is of interest since the production of the HA capsule is a very efficient and clever way that Streptococci use to evade surveillance by the immune system.
HA is synthesized by both mammalian and Streptococcus cells by the enzyme hyaluronate synthase, that has been localized to the plasma membrane of Streptococcus (Markovitz, et al., 1962). The synthesis of HA in these organisms is a multi-step process. Initiation involves binding of an initial precursor, UDP-GlcNAc or UDP-GlcA. This is followed by elongation which involves alternate addition of the two sugars to the growing oligosaccharide chain. The growing polymer is extruded across the bacterial plasma membrane region of the cell wall and into the extracellular space. Although the HA biosynthetic system was one of the first membrane heteropolysaccharide synthetic pathways studied, the mechanism of HA synthesis is still not understood. This may be because in vitro systems developed to date are inadequate in that de novo biosynthesis of HA has not been accomplished. Chain elongation but not new chain initiation occurs.
The direction of HA polymer growth is a matter of disagreement. Addition of the monosaccharides could be to the reducing (Prehm, 1983) or nonreducing (Stoolmiller, et al., 1969) end of the growing HA chain. In addition, other questions that need to be addressed are (i) whether nascent chains are linked covalently to a protein, to UDP or to a lipid intermediate, (ii) whether chains are initiated using a primer, and (iii) the mechanism by which the mature polymer is extruded through the plasma membrane of the Streptococcus. Understanding the mechanism of HA biosynthesis may allow development of alternative strategies to control Streptococcal infections by interfering in the process.
Group C S. equisimilis strain D181 synthesizes and secretes HA. Investigators have used this strain and group A strains, such as A111, to study the biosynthesis of HA and to characterize the HA-synthesizing activity in terms of its divalent cation requirement (Stoolmiller, et al., 1969), precursor (UDP-GlcNAc and UDP-GlcUA) utilization (Ishimoto, et al., 1967: Markovitz, et al., 1959), and optimum pH (Stoolmiller, et al., 1969). The HA synthase enzyme has been studied for approximately 30 years, but has not yet been identified or purified. Although a 52-kD protein has been putatively suggested as the HA synthase (Prehm, et al., 1986), this report is now known to be in error. Furthermore, no one has successfully purified to homogeneity an active enzyme. Moreover, it""s not clear whether a bona fide HA synthase molecule is all that is needed for the generation of hyaluronic acid, or whether it might act in concert with other cellular components or subunits. Thus, totally ex vivo methods of producing HA have not been forthcoming.
Typically, HA has been prepared commercially by isolation from either rooster combs or extracellular media from Streptococcal cultures. One method which has been developed for preparing HA is through the use of cultures of HA-producing streptococcal bacteria. U.S. Pat. No. 4,517,295, describes such a procedure, wherein HA-producing Streptococci are fermented under anaerobic conditions in a CO2-enriched growth medium. Under these conditions, HA is produced and can be extracted from the broth. It is generally felt that isolation of HA from rooster comb is laborious and difficult, since one starts with HA in a less pure state. The advantage of isolation from rooster comb is that the HA produced is of higher molecular weight. However, preparation of HA by bacterial fermentation is easier, since the HA is of higher purity to start with. Usually, however, the molecular weight of HA produced in this way is smaller than that from rooster combs. Therefore, a technique that would allow the production of high molecular weight HA by bacterial fermentation would be an improvement over existing procedures.
High molecular weight HA has a wide variety of useful applicationsxe2x80x94ranging from cosmetics to eye surgery (Laurent and Fraser, 1992). Due to its potential for high viscosity and its high biocompatibility, HA finds particular application in eye surgery as a replacement for vitreous fluid. HA has also been used to treat racehorses for traumatic arthritis by intra-articular injections of HA, in shaving cream as a lubricant, and in a variety of cosmetic products due to its physiochemical properties of high viscosity and its ability to retain moisture for long periods of time. In general, the higher molecular weight the HA that is employed the better. This is because HA solution viscosity increases with the average molecular weight of the individual HA polymer molecules in the solution. Unfortunately, very high molecular weight HA, such as that ranging up to 107, has been difficult to obtain by currently available isolation procedures.
To address these or other difficulties, there is a need for new methods and constructs that can be used to produce HA having one or more improved properties such as greater purity or ease of preparation. In particular, there is a need to develop methodology for the production of larger amounts of relatively higher molecular weight and purity HA than is available from current technology. The present invention addresses one or more shortcomings in the art through the application of recombinant DNA technology.
The present invention involves the application of recombinant DNA technology to solving one or more problems in the art of hyaluronic acid preparation. These problems are addressed through the isolation and use of a DNA segment encoding all or a portion of the hyaluronate synthase gene, the gene responsible for HA chain biosynthesis. The gene was cloned from DNA of an appropriate microbial source and engineered into useful recombinant constructs for the preparation of HA and for the preparation of large quantities of the HA synthase enzyme itself.
The present invention, in a general and overall sense, concerns the isolation and characterization of a hyaluronate or hyaluronic acid synthase gene, as may be used for the polymerization of glucuronic acid and N-acetylglucosamine into the glycosaminoglycan hyaluronic acid. The present inventors have identified the hasA locus and have determined the sequence encoding the Hyaluronic acid synthase (HA synthase) gene from Streptococcus. The hasA gene product, HasA, has been expressed in homologous and heterologous cells, can be used to isolate hyaluronic acid synthase, and can be used for the production of hyaluronic acid. The hasA gene also provides a new probe to assess the potential of bacterial specimens to produce hyaluronic acid.
The present invention encompasses a novel gene, hasA. The expression of this gene correlates with virulence of Streptococcal strains, by providing a means of escaping immune surveillance. The term, xe2x80x9chyaluronic acid synthasexe2x80x9d, xe2x80x9chyaluronate synthasexe2x80x9d, xe2x80x9chyaluronan synthasexe2x80x9d and xe2x80x9cHA synthasexe2x80x9d, are used interchangeably to describe an enzyme that polymerizes a glycosaminoglycan polysaccharide chain composed of alternating glucuronic acid and N-acetylglucosamine sugars.
Through the application of techniques and knowledge set forth herein, those of skill in the art will be able to obtain nucleic acid segments encoding an HA synthase gene. Through isolation of the HA gene, from whatever source is chosen, one will have the ability to realize significant advantages such as an ability to manipulate the host that is chosen to express the HA synthase gene, the fermentation environment chosen for HA production, as well as genetic manipulation of the underlying gene. As those of skill in the art will recognize, in light of the present disclosure, this will provide additional significant advantages both in the ability to control the expression of the gene and in the nature of the gene product that is realized.
Accordingly, the invention is directed to the isolation of DNA that comprises the HA synthase gene, whether it be from prokaryotic or eukaryotic sources. This is possible because the enzyme, and indeed the gene, is one found in both eukaryotes and some prokaryotes. Typical prokaryotic sources will include Group A or Group C Streptococcus sources such as S. pyogenes, S. equisimilis, or S. zooepidemicus. Eukaryotes are also known to produce HA (Ng and Schwartz, 1989) and thus have HA synthase genes that may be employed in connection with the invention. For example, it is known that HA is produced in rooster combs by mesodermal cells of the rooster. These cells can be employed to isolate starting mRNA for the production of cDNA libraries by well known techniques, which can subsequently be screened by novel screening techniques set forth herein. Other eukaryotic sources that can be employed include synovial chondrocytes and fibroblasts, dermal fibroblasts, and even trabecular-meshwork cells of the eye.
HA synthase-encoding nucleic acid segments of the present invention are defined as being isolated free of total chromosomal or genomic DNA such that they may be readily manipulated by recombinant DNA techniques. Accordingly, as used herein, the phrase xe2x80x9csubstantially purified DNA segmentxe2x80x9d refers to a DNA segment isolated free of total chromosomal or genomic DNA and retained in a state rendering it useful for the practice of recombinant techniques, such as DNA in the form of a discrete isolated DNA fragment, or a vector (e.g., plasmid, phage or virus) incorporating such a fragment.
A preferred embodiment of the present invention is a purified nucleic acid segment encoding HA synthase, wherein the segment encodes a protein having an amino acid sequence in accordance with SEQ ID NO:2, or that is capable of hybridizing to the nucleotide sequence of SEQ ID NO:1 under standard hybridization conditions as described herein. The nucleotide segment of the present invention is a purified nucleic acid segment, further defined as including a nucleotide sequence as shown in FIG. 7, and in accordance with SEQ ID NO:1.
In a more preferred embodiment the purified nucleic acid segment consists essentially of the nucleotide sequence of SEQ ID NO:1. As used herein, the term xe2x80x9cnucleic acid segmentxe2x80x9d and xe2x80x9cDNA segmentxe2x80x9d are used interchangeably and refer to a DNA molecule which has been isolated free of total genomic DNA of a particular species. Therefore, a xe2x80x9cpurifiedxe2x80x9d DNA or nucleic acid segment as used herein, refers to a DNA segment which contains an hasA coding sequence yet is isolated away from, or purified free from, total genomic DNA, for example, total Streptococcus pyogenes or, for example, mammalian host genomic DNA. Included within the term xe2x80x9cDNA segmentxe2x80x9d, are DNA segments and smaller fragments of such segments, and also recombinant vectors, including, for example, plasmids, cosmids, phage, viruses, and the like.
Similarly, a DNA segment comprising an isolated or purified hasA gene refers to a DNA segment including hasA coding sequences isolated substantially away from other naturally occurring genes or protein encoding sequences. In this respect, the term xe2x80x9cgenexe2x80x9d is used for simplicity to refer to a functional protein, polypeptide or peptide encoding unit. As will be understood by those in the art, this functional term includes genomic sequences, cDNA sequences or combinations thereof. xe2x80x9cIsolated substantially away from other coding sequencesxe2x80x9d means that the gene of interest, in this case hasA, forms the significant part of the coding region of the DNA segment, and that the DNA segment does not contain large portions of naturally-occurring coding DNA, such as large chromosomal fragments or other functional genes or DNA coding regions. Of course, this refers to the DNA segment as originally isolated, and does not exclude genes or coding regions later added to, or intentionally left in the segment by the hand of man.
Due to certain advantages associated with the use of prokaryotic sources, one will likely realize the most advantages upon isolation of the HA synthase gene from prokaryotes such as S. pyogenes or S. equisimilis. One such advantage is that, typically, eukaryotic enzymes may require significant post-translational modifications that can only be achieved in a eukaryotic host. This will tend to limit the applicability of any eukaryotic HA synthase gene that is obtained. Moreover, those of skill will likely realize additional advantages in terms of time and ease of genetic manipulation where a prokaryotic enzyme gene is sought to be employed. These additional advantages include (a) the ease of isolation of a prokaryotic gene because of the relatively small size of the genome and, therefore, the reduced amount of screening of the corresponding genomic library and (b) the ease of manipulation because the overall size of the coding region of a prokaryotic gene is significantly smaller due to the absence of introns. Furthermore, if the product of the HA synthase gene (i.e., the enzyme) requires posttranslational modifications, these would best be achieved in a similar prokaryotic cellular environment (host) from which the gene was derived.
Preferably, DNA sequences in accordance with the present invention will further include genetic control regions which allow the expression of the sequence in a selected recombinant host. Of course, the nature of the control region employed will generally vary depending on the particular use (e.g., cloning host) envisioned. For example, in streptococcal hosts, the preferred control region is the homologous control region associated with the structural gene in its natural state. The homologous control region, in fact, may be coisolated directly with the isolation of the HA synthase structural gene itself through the practice of certain preferred techniques disclosed herein.
In particular embodiments, the invention concerns isolated DNA segments and recombinant vectors incorporating DNA sequences which encode an hasA gene, that includes within its amino acid sequence an amino acid sequence in accordance with SEQ ID NO:2. Moreover, in other particular embodiments, the invention concerns isolated DNA segments and recombinant vectors incorporating DNA sequences which encode a gene that includes within its amino acid sequence the amino acid sequence of an hasA gene corresponding to Streptococcus pyogenes hasA. Naturally, where the DNA segment or vector encodes a full length HasA protein, or is intended for use in expressing the HasA protein, the most preferred sequences are those which are essentially as set forth in SEQ ID NO:2.
Nucleic acid segments having HA synthase activity may be isolated by the methods described hereinabove. The term xe2x80x9ca sequence essentially as set forth in SEQ ID NO:2xe2x80x9d means that the sequence substantially corresponds to a portion of SEQ ID NO:2 and has relatively few amino acids which are not identical to, or a biologically functional equivalent of, the amino acids of SEQ ID NO:2. The term xe2x80x9cbiologically functional equivalentxe2x80x9d is well understood in the art and is further defined in detail herein, as a gene having a sequence essentially as set forth in SEQ ID NO:2, and that is associated with the ability of Streptococcus to produce HA and a hyaluronic acid coat. Accordingly, sequences which have between about 70% and about 80%; or more preferably, between about 81% and about 90%; or even more preferably, between about 91% and about 99%; of amino acids which are identical or functionally equivalent to the amino acids of SEQ ID NO:2 will be sequences which are xe2x80x9cessentially as set forth in SEQ ID NO:2xe2x80x9d.
Another preferred embodiment of the present invention is a purified nucleic acid segment that encodes a protein in accordance with SEQ ID NO:2, further defined as a recombinant vector. As used herein the term, xe2x80x9crecombinant vectorxe2x80x9d, refers to a vector that has been modified to contain a nucleic acid segment that encodes an HasA protein, or fragment thereof. The recombinant vector may be further defined as an expression vector comprising a promoter operatively linked to said HasA encoding nucleic acid segment.
A further preferred embodiment of the present invention is a host cell, made recombinant with a recombinant vector comprising an hasA gene. The preferred recombinant host cell may be a prokaryotic cell. In another embodiment, the recombinant host cell is a eukaryotic cell. As used herein, the term xe2x80x9cengineeredxe2x80x9d or xe2x80x9crecombinantxe2x80x9d cell is intended to refer to a cell into which a recombinant gene, such as a gene encoding hasA, has been introduced. Therefore, engineered cells are distinguishable from naturally occurring cells which do not contain a recombinantly introduced gene. Engineered cells are thus cells having a gene or genes introduced through the hand of man. Recombinantly introduced genes will either be in the form of a cDNA gene, a copy of a genomic gene, or will include genes positioned adjacent to a promoter not naturally associated with the particular introduced gene.
Where one desires to use a host other than Streptococcus, as may be used to produce recombinant HA synthase, it may be advantageous to employ a prokaryotic system such as E. coli, B. subtilis, Lactococcus sp., or even eukaryotic systems such as yeast or Chinese hamster ovary, African green monkey kidney cells, VERO cells, or the like. Of course, where this is undertaken, it will generally be desirable to bring the HA synthase gene under the control of sequences which are functional in the selected alternative host. The appropriate DNA control sequences, as well as their construction and use, are generally well known in the art as discussed in more detail herein below.
In preferred embodiments, the HA synthase-encoding DNA segments further include DNA sequences, known in the art functionally as origins of replication or xe2x80x9crepliconsxe2x80x9d, which allow replication of contiguous sequences by the particular host. Such origins allow the preparation of extrachromosomally localized and replicating chimeric segments or plasmids, to which HA synthase DNA sequences are ligated. In more preferred instances, the employed origin is one capable of replication in Streptococcus hosts. However, for more versatility of cloned DNA segments, it may be desirable to alternatively or even additionally employ origins recognized by other host systems whose use is contemplated (such as in a shuttle vector).
The isolation and use of other replication origins such as the SV40, polyoma or bovine papilloma virus origins, which may be employed for cloning in a number of higher organisms, are well known (Fiers, et al., 1978). In certain embodiments, the invention may thus be defined in terms of a recombinant transformation vector which includes the HA synthase gene sequence together with an appropriate replication origin and under the control of selected control regions.
In accordance with the present invention, the HA synthase gene, when from a prokaryotic source such as a Streptococcal source, is obtained by the following general steps. First, the genetic loci are identified by transposon insertional mutagenesis. One such transposon system is the TN916, obtainable from the transposon donor strain E. faecalis CG110, which was used to mutate the mucoid strain of Streptococcus pyogenes S43. Mutants were isolated and the genomic DNA surrounding the transposon was sequenced and used to derive oligonueleotides for use in cloning the wild-type gene. Phage libraries were screened, and two clones, xcex1X and xcex2Y, were obtained that contained the predicted sequence. The locus was characterized by restriction mapping and southern blot analysis.
Thus, although the present invention is exemplified in terms of clones screened via transposon mediated mutagenesis, it will be appreciated by those of skill in the art that other means may be used to obtain the hasA gene, in light of the present disclosure. For example, polymerase chain reaction produced DNA fragments may be obtained which contain full complements of genes from a number of sources, including other strains of Streptococcus or from eukaryotic sources, such as cDNA libraries. Virtually any molecular cloning approach may be employed for the generation of DNA fragments in accordance with the present invention. Thus, the only limitation generally on the particular method employed for DNA isolation is that the isolated nucleic acids should encode a biologically functional equivalent HA synthase.
Once the DNA has been isolated it is ligated together with a selected vector. Virtually any cloning vector can be employed to realize advantages in accordance with the invention. Typical useful vectors include plasmids and phages for use in prokaryotic organisms and even viral vectors for use in eukaryotic organisms. Examples include pBluescript(trademark), pSA3, lambda, SV40, polyoma, adenovirus, bovine papilloma virus and retroviruses. However, it is believed that particular advantages will ultimately be realized where vectors capable of replication in both Lactococcus or Bacillus strains and E. coli are employed.
Vectors such as these, exemplified by the pSA3 vector of Dao and Ferretti (Dao, et al., 1985) or the pAT19 vector of Trieu-Cuot, et al. (1991), allow one to perform clonal colony selection in an easily manipulated host such as E. coli, followed by subsequent transfer back into a Lactococcus or Bacillus strain for production of HA. This is advantageous in that one can augment the Lactococcus or Bacillus strain""s ability to synthesize HA through gene dosaging (i.e., providing extra copies of the HA synthase gene by amplification) and/or inclusion of additional genes to increase the availability of HA precursors. The inherent ability of Streptococci to synthesize HA can also be augmented through the formation of extra copies, or amplification, of the plasmid that carries the HA synthase gene. This amplification can account for up to a 10-fold increase in plasmid copy number and, therefore, the HA synthase gene copy number.
Another procedure that would further augment HA synthase gene copy number is the insertion of multiple copies of the gene into the plasmid. This extra amplification would be especially feasible, since the bacterial HA synthase gene size is small. In any event, the chromosomal DNA-ligated vector is employed to transfect the host that is selected for clonal screening purposes such as E. coli, through the use of a vector that is capable of expressing the inserted DNA in the chosen host.
Where a eukaryotic source such as dermal or synovial fibroblasts or rooster comb cells is employed, one will desire to proceed initially by preparing a cDNA library. This is carried out first by isolation of mRNA from the above cells, followed by preparation of double stranded cDNA using an enzyme with reverse transcriptase activity and ligation with the selected vector. Numerous possibilities are available and known in the art for the preparation of the double stranded cDNA, and all such techniques are believed to be applicable. A preferred technique involves reverse transcription. Once a population of double stranded cDNAs is obtained, a cDNA library is prepared in the selected host by accepted techniques, such as by ligation into the appropriate vector and amplification in the appropriate host.
Due to the high number of clones that are obtained, and the relative ease of screening large numbers of clones by the techniques set forth herein, one may desire to employ phage expression vectors, such as xcexgt11 or xcexgt12, for the cloning and expression screening of cDNA clones.
Due to the general absence of correct information regarding the HA synthase enzyme, traditional approaches to clonal screening, such as oligonucleotide hybridization or immunological screening, was not available. Accordingly, it was necessary for the inventors to use an alternative approach based on phenotype to screen and select for the HA synthase that rely on abrogating expression of HA synthase activity. The methods which were developed can be applied to screen the selected host, regardless of whether a eukaryotic or prokaryotic gene is sought. One method involves the application of a dye exclusion technique to identify clones which contain HA. The typical dye employed, India ink, is excluded from an HA capsule and allows visualization of HA by negative staining. A second method involves positive staining, such as with Alcian Blue, to identify HA producing clones. Alcian Blue binds to and stains polyanionic molecules such as HA (Scott, et al., 1964). However, in that India ink or Alcian Blue is not entirely specific for HA, the present inventors employed additional screening methods as described in Examples I and II.
A variety of additional screening and validation procedures are also set forth herein that can variously be employed to identify the presence of either the HA enzyme or its HA product as a means for identifying positive clones or negative clones (mutants). These procedures included the use of Percoll gradient centrifugation and the ability of membrane fractions from candidate clones to incorporate authentic radiolabeled sugar nucleotides into high molecular weight HA.
In certain other embodiments, the invention concerns isolated DNA segments and recombinant vectors that include within their sequence a nucleic acid sequence essentially as set forth in SEQ ID NO:1. The term xe2x80x9cessentially as set forth in SEQ ID NO:1xe2x80x9d, is used in the same sense as described above and means that the nucleic acid sequence substantially corresponds to a portion of SEQ ID NO:1, and has relatively few codons which are not identical, or functionally equivalent, to the codons of SEQ ID NO:1. The term xe2x80x9cfunctionally equivalent codonxe2x80x9d is used herein to refer to codons that encode the same amino acid, such as the six codons for arginine or serine, as set forth in Table I, and also refers to codons that encode biologically equivalent amino acids.
It will also be understood that amino acid and nucleic acid sequences may include additional residues, such as additional N- or C-terminal amino acids or 5xe2x80x2 or 3xe2x80x2 sequences, and yet still be essentially as set forth in one of the sequences disclosed herein, so long as the sequence meets the criteria set forth above, including the maintenance of biological protein activity where protein expression and enzyme activity is concerned. The addition of terminal sequences particularly applies to nucleic acid sequences which may, for example, include various non-coding sequences flanking either of the 5xe2x80x2 or 3xe2x80x2 portions of the coding region or may include various internal sequences, which are known to occur within genes.
Allowing for the degeneracy of the genetic code, sequences which have between about 70% and about 80%; or more preferably, between about 80% and about 90%; or even more preferably, between about 90% and about 99%; of nucleotides which are identical to the nucleotides of SEQ ID NO:1 will be sequences which are xe2x80x9cessentially as set forth in SEQ ID NO:1xe2x80x9d. Sequences which are essentially the same as those set forth in SEQ ID NO:1 may also be functionally defined as sequences which are capable of hybridizing to a nucleic acid segment containing the complement of SEQ ID NO:1 under relatively stringent conditions. Suitable relatively stringent hybridization conditions will be well known to those of skill in the art and are clearly set forth herein, for example conditions for use with southern and northern blot analysis.
The term xe2x80x9cstandard hybridization conditionsxe2x80x9d as used herein, is used to describe those conditions under which substantially complementary nucleic acid segments will form standard Watson-Crick base-pairing. A number of factor are known that determine the specificity of binding or hybridization, such as pH, salt concentration, the presence of chaotropic agents (e.g. formamide and dimethyl sulfoxide)., the length of the segments that are hybridizing, and the like.
For use with the present invention, standard hybridization conditions for relatively large segments, that is segments longer than about 100 nucleotides, will include a hybridization mixture having between about 0.3 to 0.6 M NaCl, a divalent cation chelator (e.g. EDTA at about 0.05 mM to about 0.5 mM), and a buffering agent (e.g. Na2PO4 at about 10 mM to 100 mM, pH 7.2), at a temperature of about 65xc2x0 C. The preferred conditions for hybridization are a hybridization mixture comprising 0.5 M NaCl, 5 mM EDTA, 0.1 M Na2PO4, pH 7.2 and 1% N-lauryl sarcosine, at a temperature of 65xc2x0 C. Naturally, conditions that affect the hybridization temperature, such as the addition of chaotropic agents, such as formamide, will be known to those of skill in the art, and are encompassed by the present invention.
When it is contemplated that shorter nucleic acid segments will be used for hybridization, for example fragments between about 14 and about 100 nucleotides, salt and temperature conditions will be altered to increase the specificity of nucleic acid segment binding. Preferred conditions for the hybridization of short nucleic acid segments include lowering the hybridization temperature to about 37xc2x0 C., and increasing the salt concentration to about 0.5 to 1.5 M NaCl with 1.5 M NaCl being particularly preferred.
Naturally, the present invention also encompasses DNA segments which are complementary, or essentially complementary, to the sequence set forth in SEQ ID NO:1. Nucleic acid sequences which are xe2x80x9ccomplementaryxe2x80x9d are those which are capable of base-pairing according to the standard Watson-Crick complementarity rules. As used herein, the term xe2x80x9ccomplementary sequencesxe2x80x9d means nucleic acid sequences which are substantially complementary, as may be assessed by the same nucleotide comparison set forth above, or as defined as being capable of hybridizing to the nucleic acid segment of SEQ ID NO:1 under relatively stringent conditions such as those described herein in Example III.
The nucleic acid segments of the present invention, regardless of the length of the coding sequence itself, may be combined with other DNA sequences, such as promoters, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, other coding segments, and the like, such that their overall length may vary considerably. It is therefore contemplated that a nucleic acid fragment of almost any length may be employed, with the total length preferably being limited by the ease of preparation and use in the intended recombinant DNA protocol. For example, nucleic acid fragments may be prepared which include a short stretch complementary to SEQ ID NO:1, such as about 10 to 15 or 20, 30, or 40 or so nucleotides, and which are up to 10,000 or 5,000 base pairs in length, with segments of 3,000 being preferred in certain cases. DNA segments with total lengths of about 1,000, 500, 200, 100 and about 50 base pairs in length are also contemplated to be useful.
A preferred embodiment of the present invention is a nucleic acid segment which comprises at least a 10-14 nucleotide long stretch which corresponds to, or is complementary to, the nucleic acid sequence of SEQ ID NO:1. In a more preferred embodiment the nucleic acid is further defined as comprising at least a 20 nucleotide long stretch, a 30 nucleotide long stretch, 50 nucleotide long stretch, 100 nucleotide long stretch, a 200 nucleotide long stretch, a 500 nucleotide long stretch, a 1000 nucleotide long stretch, a 1500 nucleotide long stretch, or at least a 1441 nucleotide long stretch which corresponds to, or is complementary to, the nucleic acid sequence of SEQ ID NO:1. The nucleic acid segment may be further defined as having the nucleic acid sequence of SEQ ID NO:1.
A related embodiment of the present invention is a nucleic acid segment which comprises at least a 10-14 nucleotide long stretch which corresponds to, or is complementary to, the nucleic acid sequence of SEQ ID NO:1, further defined as comprising a nucleic acid fragment of up to 10,000 basepairs in length. A more preferred embodiment if a nucleic acid fragment comprising from 14 nucleotides of SEQ ID NO:1 up to 5,000 basepairs in length, 3,000 basepairs in length, 1,000 basepairs in length, 500 basepairs in length, or 100 basepairs in length.
Naturally, it will also be understood that this invention is not limited to the particular nucleic acid and amino acid sequences of SEQ ID NOS:1 and 2. Recombinant vectors and isolated DNA segments may therefore variously include the hasA coding regions themselves, coding regions bearing selected alterations or modifications in the basic coding region, or they may encode larger polypeptides which nevertheless include hasA-coding regions or may encode biologically functional equivalent proteins or peptides which have variant amino acids sequences.
The DNA segments of the present invention encompass biologically functional equivalent HasA proteins and peptides. Such sequences may arise as a consequence of codon redundancy and functional equivalency which are known to occur naturally within nucleic acid sequences and the proteins thus encoded. Alternatively, functionally equivalent proteins or peptides may be created via the application of recombinant DNA technology, in which changes in the protein structure may be engineered, based on considerations of the properties of the amino acids being exchanged. Changes designed by man may be introduced through the application of site-directed mutagenesis techniques e.g., to introduce improvements to the enzyme activity or to antigenicity of the HasA protein or to test HasA mutants in order to examine HA synthase activity at the molecular level.
A preferred embodiment of the present invention is a purified composition comprising a polypeptide having an amino acid sequence in accordance with SEQ ID NO:2. The term xe2x80x9cpurifiedxe2x80x9d as used herein, is intended to refer to an HasA protein composition, wherein the HasA protein is purified to any degree relative to its naturally-obtainable state, i.e., in this case, relative to its purity within a prokaryotic cell extract. A preferred cell for the isolation of HasA protein is a Streptococcus pyogenes cell, however, HasA protein may also be isolated from other members of the Streptococcus genus, patient specimens, recombinant cells, infected tissues, isolated subpopulation of tissues that contain high levels of hyaluronate in the extracellular matrix, and the like, as will be known to those of skill in the art, in light of the present disclosure. A purified HasA protein composition therefore also refers to a polypeptide having the amino acid sequence of SEQ ID NO:2, free from the environment in which it may naturally occur.
If desired, one may also prepare fusion proteins and peptides, e.g., where the HasA protein coding regions are aligned within the same expression unit with other proteins or peptides having desired functions, such as for purification or immunodetection purposes (e.g., proteins which may be purified by affinity chromatography and enzyme label coding regions, respectively).
Turning to the expression of the hasA gene whether from genomic DNA, or a cDNA one may proceed to prepare an expression system for the recombinant preparation of HasA protein. The engineering of DNA segment(s) for expression in a prokaryotic or eukaryotic system may be performed by techniques generally known to those of skill in recombinant expression. For example, one may prepare a HasA-GST (glutathione-S-transferase) fusion protein that is a convenient means of bacterial expression. However, it is believed that virtually any expression system may be employed in the expression of HasA.
HasA may be successfully expressed in eukaryotic expression systems, however, the inventors aver that bacterial expression systems can be used for the preparation of HasA for all purposes. The cDNA for HasA may be separately expressed in bacterial systems, with the encoded proteins being expressed as fusions with xcex2-galactosidase, avidin, ubiquitin, Schistosoma japonicum glutathione S-transferase, maltose-binding protein, polyhistidine-tags, epitope-tags (e.g., myc and FLAG) and the like. It is believed that bacterial expression will ultimately have advantages over eukaryotic expression in terms of ease of use and quantity of materials obtained thereby.
It is proposed that transformation of host cells with DNA segments encoding HasA will provide a convenient means for obtaining an HasA protein. It is also proposed that cDNA, genomic sequences, and combinations thereof, are suitable for eukaryotic expression, as the host cell will, of course, process the genomic transcripts to yield functional mRNA for translation into protein.
Another embodiment of the present invention is a method of preparing a protein composition comprising growing recombinant host cell comprising a vector that encodes a protein which includes an amino acid sequence in accordance with SEQ ID NO:2. The host cell will be grown under conditions permitting nucleic acid expression and protein production followed by recovery of the protein so produced. The production of HA synthase and HA, including: the host cell, conditions permitting nucleic acid expression, protein production and recovery will be known to those of skill in the art in light of the present disclosure of the hasA gene, and the hasA gene protein product HasA, and by the methods described in Examples III, IV, and V.
Preferred hosts for the expression of hyaluronic acid are prokaryotes, such as S. pyogenes, S. equisimilis, and other suitable members of the Streptococcus species. However, it is also contemplated that HA may be synthesized by heterologous host cells expressing HA synthase, such as species members of the Bacillus, Salmonella, Pseudomonas, Enterococcus, or even Escherichia genus. A most preferred host for expression of the HA synthase of the present invention is a bacteria transformed with the hasA gene of the present invention, such as Lactococcus, Bacillus subtilis or S. pyogenes. 
It is similarly believed that almost any eukaryotic expression system may be utilized for the expression of hasA e.g., baculovirus-based, glutamine synthase-based, dihydrofolate reductase-based systems, SV-40 based, adenovirus-based, cytomegalovirus-based, and the like, could be employed. For expression in this manner, one would position the coding sequences adjacent to and under the control of the promoter. It is understood in the art that to bring a coding sequence under the control of such a promoter, one positions the 5xe2x80x2 end of the transcription initiation site of the transcriptional reading frame of the protein between about 1 and about 50 nucleotides xe2x80x9cdownstreamxe2x80x9d of (i.e., 3xe2x80x2 of) the chosen promoter.
Where eukaryotic expression is contemplated, one will also typically desire to incorporate into the transcriptional unit which includes the hasA gene, an appropriate polyadenylation site (e.g., 5xe2x80x2 -AATAAA-3xe2x80x2) if one was not contained within the original cloned segment. Typically, the poly A addition site is placed about 30 to 2000 nucleotides xe2x80x9cdownstreamxe2x80x9d of the termination site of the protein at a position prior to transcription termination.
It is contemplated that virtually any of the commonly employed host cells can be used in connection with the expression of hasA in accordance herewith. Examples of preferred cell lines for expressing the HA synthase gene of the present invention include cell lines typically employed for eukaryotic expression such as 239, AtT-20, HepG2, VERO, HeLa, CHO, WI 38, BHK, COS-7, RIN and MDCK cell lines.
This will generally include the steps of providing a recombinant host bearing the recombinant DNA segment encoding the HA synthase enzyme and capable of expressing the enzyme; culturing the recombinant host in media under conditions that will allow for transcription of the cloned HA gene and appropriate for the production of the hyaluronic acid; and separating and purifying the HA synthase enzyme or the secreted hyaluronic acid from the recombinant host.
Generally, the conditions appropriate for expression of the cloned HA synthase gene will depend upon the promoter, the vector, and the host system that is employed. For example, where one employs the lac promoter, one will desire to induce transcription through the inclusion of a material that will stimulate lac transcription, such as IPTG. Where other promoters are employed, different materials may be needed to induce or otherwise up-regulate transcription. In addition, to obtaining expression of the synthase, one will preferably desire to provide an environment that is conducive to HA synthesis by including appropriate genes encoding enzymes needed for the biosynthesis of sugar nucleotide precursors, and by using growth media containing substrates for the precursor-supplying enzymes, such N-acetylglucosamine (GlcNAc) and glucose (Glc).
One may further desire to incorporate the gene in a host which is defective in the enzyme hyaluronidase, so that the product synthesized by the enzyme will not be degraded in the medium. Furthermore, a host would be chosen to optimize production of HA. For example, a suitable host would be one that produced large quantities of the sugar nucleotide precursors to support the HA synthase enzyme and allow it to produce large quantities of HA. Such a host may be found naturally or may be made by a variety of techniques including mutagenesis or recombinant DNA technology. The genes for the sugar nucleotide synthesizing enzymes, particularly the UDP-Glc dehydrogenase required to produce UDP-GlcA, could also be isolated and incorporated in a vector along with the HA synthase gene. A preferred embodiment of the present invention is a host containing these ancillary recombinant genes and the amplification of these gene products thereby allowing for increased production of HA.
In the case where production of HA synthase is desired, the enzyme is preferably synthesized in bacteria using the T7 expression system (Studier, et al., 1990). pT5 plasmids containing the HA synthase gene inserted adjacent to the philo promoter are transformed into E. coli stain BL21(DE3)pLysS. In this strain the T7 gene encoding the bacteriophage RNA polymerase is under control of the E. coli lacZ promoter. Therefore, the polymerase can be induced by IPTG and transcription of the HA synthase gene is, in turn, induced from the xcfx8610 promoter of the pT5 vector.
The means employed for culturing of the host cell is not believed to be particularly crucial. For useful details, one may wish to refer to the disclosure of U.S. Pat. Nos. 4,517,295; 4,801,539; 4,784,990: or 4,780,414: all incorporated herein by reference. Where a prokaryotic host is employed, such as S. pyogenes or S. equisimilis, one may desire to employ a fermentation of the bacteria under anaerobic conditions in CO2-enriched broth growth media. This allows for a greater production of HA than under aerobic conditions. Another consideration is that Streptococcal cells grown anaerobically do not produce pyrogenic exotoxins. Appropriate growth conditions can be customized for other prokaryotic hosts, as will be known to those of skill in the art, in light of the present disclosure.
Once the appropriate host has been constructed, and cultured under conditions appropriate for the production of HA, one will desire to separate the HA so produced. Typically, the HA will be secreted or otherwise shed by the recombinant organism into the surrounding media, allowing the ready isolation of HA from the media by known techniques. For example, HA can be separated from the media by filtering and/or in combination with precipitation by alcohols such as ethanol. Other precipitation agents include organic solvents such as acetone or quaternary organic ammonium salts such as cetyl pyridinium chloride (CPC).
A preferred technique for isolation of HA is described in U.S. Pat. No. 4,517,295 in which the organic carboxylic acid, trichloroacetic acid, is added to the bacterial suspension at the end of the fermentation. The trichloroacetic acid causes the bacterial cells to clump and die and facilitates the ease of separating these cells and associated debris from HA, the desired product. The clarified supernatant is concentrated and dialyzed to remove low molecular weight contaminants including the organic acid. The aforementioned procedure utilizes Millipore(tm) filtration through filter cassettes containing 0.22 xcexcm pore size filters. Diafiltration is continued until the conductivity of the solution decreases to approximately 0.5 mega-ohms.
The concentrated HA is precipitated by adding an excess of reagent grade ethanol or other organic solvent and the precipitated HA is then dried by washing with ethanol and vacuum dried, lyophilized or spray dried to remove alcohol. The HA can then be redissolved in a borate buffer, pH 8, and precipitated with CPC or certain other organic ammonium salts such as CETAB, a mixed trimethyl ammonium bromide solution at 4 degree(s) C. The precipitated HA is recovered by coarse filtration, resuspended in 1 M NaCl, diafiltered and concentrated as further described in the above referenced patent. The resultant HA is filter sterilized and ready to be converted to an appropriate salt, dry powder or sterile solution, depending on the desired end use.